The present invention relates to the fields of molecular genetics and diagnostics.
Many diseases linked to genome modifications, either of the host organism or of infectious organisms, are often the consequence of a change in a small number of nucleotides, frequently involving a change in a single nucleotide. Such single nucleotide changes are referred to as single nucleotide polymorphisms or simply SNPs, and the site at which the SNP occurs is typically referred to as a polymorphic site.
The ability to detect specific nucleotide alterations or mutations in DNA sequences is an important medical tool. The ability to identify such alterations provides a means for diagnosing many common diseases that are associated with SNPs, such as diabetes, thalassemia, sickle-cell anemia, cystic fibrosis, and certain oncogenic mutations. Methods capable of quickly identifying such changes or mutations are also valuable in taking prophylactic measures, assessing the propensity for disease and inpatient counseling and education.
Methods for determining which nucleotide is present at a polymorphic site are sometimes referred to as genotyping. Several methods are available for genotyping SNPs, including allele-specific oligonucleotide hybridization, mini-sequencing or primer extension, the TaqMan assay, and the oligo-ligation assay. However, frequently these methods include one or more shortcomings such as high cost, relatively long analysis times, use of radiolabeled reagents, complexity and/or being poorly suited for multiplex analysis wherein multiple analyses are conducted at the same time.
The present invention provides various methods and kits for determining the identity of a nucleotide at a variant site such as a polymorphism, for example. For instance, certain methods of the invention involve analyzing a variant site at which a first or second nucleotide in a target nucleic acid is located. Such methods include amplifying the target nucleic acid utilizing a first and second primer, wherein the first primer is complementary to a segment of a first strand of the target nucleic acid and the 3xe2x80x2 end of the primer is adjacent to, but does not overlap, the variant site. The second primer is complementary to a segment of a second strand of the target nucleic acid and includes a nucleotide derivative resistant to digestion by a 5xe2x80x2-3xe2x80x2 exonuclease. The first and second primers flank the variant site once they have hybridized to their respective strands.
Template-dependent extension of the first and second primers in the presence of four deoxynucleoside triphosphates (dATP, dTTP, dGTP and dCTP) is then conducted, wherein one of the deoxynucleoside triphosphates is an analog of a natural deoxynucleoside triphosphate that is resistant to digestion by 5xe2x80x2-3xe2x80x2 exonucleases (e.g., a thiol base, a phosphorylated base or a boronated base). This deoxynucleotide analog is selected to be the complement of the first or second base at the variant site in the second strand and the target nucleic acid serves as a template such that an amplified double-stranded product is generated. The resulting double-stranded product is then digested with the 5xe2x80x2-3xe2x80x2 exonuclease (e.g., phage T7 gene 6 exonuclease or lambda nuclease) to form a digested product having a single-stranded segment. The single-stranded segment in turn is removed with an enzyme that digests single-stranded DNA to produce a blunt end fragment. The size of the blunt-end fragment is then determined as an indicator of whether the variant site includes the first or second nucleotide.
The invention further provides related methods for conducting a multiplex analysis of multiple variant sites at the same time. For example, the invention provides certain methods for analyzing a first and second variant site in a first and second target nucleic acid, respectively. Each variant site can include a first or second base. Such methods generally begin by amplifying the first and second target nucleic acid by providing a first upstream and downstream primer pair. The first upstream primer is complementary to a segment of a first strand of the first target nucleic acid and its 3xe2x80x2 end is adjacent to, but does not overlap the first variant site. The first downstream primer is complementary to a segment of a second strand of the first target nucleic acid and includes a nucleotide derivative resistant to digestion by a 5xe2x80x2-3xe2x80x2 exonuclease. Upon hybridization to their respective strands, the first upstream and downstream primer flank the first variant site.
A second upstream and downstream primer pair are also provided. The second upstream primer is complementary to a segment of a first strand of the second target nucleic acid and the 3xe2x80x2 end of the second upstream primer is adjacent to, but does not overlap, the second variant site. The second downstream primer is complementary to a segment of a second strand of the second target nucleic acid and includes a nucleotide derivative resistant to digestion by a 5xe2x80x2-3xe2x80x2 exonucleases. The second upstream and downstream primer flank the second variant site once they hybridize to their respective strands.
Following hybridization of the primers, template dependent extension of the first and second primer pairs is conducted in the presence of four deoxynucleoside triphosphates (dATP, dTTP, dGTP and dCTP) wherein one of the deoxynucleoside triphosphates is an analog of a natural deoxynucleoside triphosphate that is resistant to digestion by 5xe2x80x2-3xe2x80x2 exonucleases. This deoxynucleotide analog is selected to be the complement of the first or second base at the first and second variant site in the second strand of the first and second target nucleic acids. The first and second target nucleic acid each serve as a template, such that an amplified double-stranded product is generated from each of the first and second primer pairs, thereby generating a plurality of double-stranded products.
Thee plurality of double-stranded products is digested with the 5xe2x80x2-3xe2x80x2 exonuclease to form a plurality of digested products, each having a single-stranded segment which is removed with an enzyme that digests single-stranded DNA. This digestion reaction produces a plurality of blunt end fragments. The size of these fragment is determined to identify whether the first and second variant sites include the first or second base.
In another aspect, the invention provides kits for analyzing a variant site in a target nucleic acid. Certain kits of the invention include a deoxynucleotide analog for use in primer extension reactions which when incorporated into an extension product is resistant to digestion by 5xe2x80x2-3xe2x80x2 exonucleases, a 5xe2x80x2-3xe2x80x2 exonuclease and an enzyme that digests single-stranded DNA. In some kits, the deoxynucleotide analog is a thiol base, a phosphorylated base or a boronated analog. Still other kits further include an enzyme that digests single-stranded DNA (e.g., mung bean nuclease and S1 nuclease). The kits may also contain two primers. If included in the kit, one primer is complementary to a segment of a first strand in the target nucleic acid such that the 3xe2x80x2 end of the primer is adjacent to, but does not overlap, the variant site of the target. The second primer is complementary to a downstream segment of a second strand of the target nucleic acid and includes a nucleotide analog derivative that is resistant to digestion by an exonuclease.